News and Publications

Structure and Action of Molecular Machines

* University of Basel, Basel, Switzerland

Macromolecular assemblies may be composed of only a few or perhaps scores of proteins and are the functional units--the molecular machines--of the cell. We use electron cryomicroscopy and image analysis to study the structure and mechanism of action of several of these molecular machines. We combine the 3-dimensional maps calculated from electron images of the assemblies with the x-ray structures of the individual components to build models of the working machines.

In our research on myosin and kinesin motors, we used this approach to visualize the various stages in the chemomechanical cycle of the track-motor complexes. Our findings have been combined with the wealth of biochemical and biophysical data from other laboratories to provide models for the action of the most well-studied motors. Movies showing the motions of brain kinesin and conventional myosin can be viewed at www.scripps.edu/milligan/projects.html.

Although the mechanism of plus end-directed, processive motion by the conventional KinN kinesins is now well understood, the mechanism by which members of the KinC kinesins (e.g., Ncd) move toward the minus ends of microtubules is not. Likewise, in the myosin superfamily, how nucleotide-mediated conformational changes in the motor domain of class VI myosins result in "backwards" motility is not known. We are elucidating the molecular mechanisms of these more unusual members of the myosin and kinesin superfamilies.

Whereas KinN and KinC kinesins move along intact microtubules, members of the KinI kinesins depolymerize microtubules and do not appear to have motile properties. We found that a KinI fragment consisting of only the conserved motor core is necessary and sufficient for ATP-dependent depolymerization. The motor core binds along microtubules in all nucleotide states, but in the presence of a nonhydrolyzable ATP analog, depolymerization also occurs. Structural characterization of the analog-induced depolymerization products revealed a snapshot of the disassembly machine at the microtubule ends. Our data indicate that whereas conventional kinesins use the energy of ATP binding to execute a power stroke that results in unidirectional motion along the microtubule surface, KinIs use the energy to bend the underlying protofilament, thereby destabilizing the microtubule lattice and leading to microtubule depolymerization.

We recently investigated the interaction of the microtubule-associated proteins MAP2c and tau with microtubules. These microtubule-stabilizing proteins are unstructured in solution but appear to become folded when they interact with the tubulin C terminus and bind to microtubules. We showed that the proteins bind longitudinally along the outer crest of tubulin protofilaments, close to the primary binding site for microtubule motors. The longitudinal interaction geometry suggests that MAP2c and tau stabilize microtubules by bridging tubulin interfaces along the protofilament and preventing the straight-to-curled transition that results in depolymerization.

We extended our studies on VCP/p97, a member of the AAA ATPase family of proteins. This protein is involved in a wide variety of cellular processes, including organelle assembly, homotypic membrane fusion, and protein degradation. We examined VCP/p97 in various nucleotide states by using electron cryomicroscopy and single-particle image analysis. The resulting 3-dimensional maps of the hexameric protein assembly show that it undergoes substantial conformational changes during the ATPase cycle. Nucleotide-dependent rearrangements of the subunits are accompanied by constriction of the central channel opening and changes in the interaction geometry of the N-terminal domain of the protein.

We developed a general method for helical crystallization of proteins on lipid tubules, and we are using it to study the virulence factor PFO from Clostridium perfringens. PFO is a cytolysin, an important class of proteins that oligomerize and embed within membranes as part of their lytic function. We obtained helical crystals of wild-type and several mutant forms of PFO on nickel-lipid tubules. Three-dimensional maps of these proteins derived from images of the helical crystals will be used to complement our studies of PFO pore formation on lipid layers. These studies will provide a better understanding of the pathogenic function of cytolysins. Additional studies involving tubular crystallization of membrane proteins and other bacterial toxins are opening up promising new areas for future research. Finally, in collaborations with the Automated Imaging Group led by B. Carragher and C. Potter, Department of Cell Biology, we are developing and implementing automatic grid searching, image acquisition, and image analysis protocols for molecular microscopy.

Publications

Hinshaw, J.E., Milligan, R.A. Nuclear pore complexes exceeding eightfold rotational symmetry. J. Struct. Biol. 14:259, 2003.

Rouiller, I., DeLaBarre, B., May, A.P., Weiss, W.I., Brunger, A.T., Milligan, R.A., Wilson-Kubalek, E.M. Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle. Nat. Struct. Biol. 9:950, 2002.